Figure 1.
Effects of dynactin subunit depletion on mitotic progression. (A through C) Analysis of mitotic figures in asynchronous Cos7 populations stained for phospho-histone H3 (A, B) or DAPI (C). (A) Mitotic progression in cells transfected with dynactin subunit siRNAs alone or in conjunction with p27 rescue plasmids. Mitotic stages were determined on the basis of chromosome configuration (mean±s.d., three experiments, n>6000 cells). Mitotic cells exhibiting incomplete chromosome congression were scored as being in prometaphase. (B) Mitotic indices of cell populations transfected with dynactin subunit siRNAs (as in A; mean±s.d., three experiments, n>6000 cells). (C) Representative images of chromosome alignment phenotypes in metaphase (left) and anaphase (right) cells in the control and p27/p25-depleted population. Bar=5 μm. (D) Mitotic populations of cells treated with dynactin subunit siRNAs were scored for the presence of misaligned or lagging chromosomes (as in C; mean±s.d., three experiments, n>600 mitotic cells). (E) Images of living control and p27/p25-depleted cells expressing histone 2B-mCherry starting 6 min prior to nuclear envelope breakdown (0:00 time stamp) and continuing through anaphase. Arrows indicate examples of uncongressed or lagging chromosomes. Twenty-five cells were filmed in each condition, and 76% (19/25 cells) of p27/p25-depleted cells had uncongressed or lagging chromosomes. Bar=5 μm. (F) The elapsed time (mean±s.d.; n=25 cells per condition) determined from live-cell recordings (as in E and Supplementary Movies) between nuclear envelope breakdown and anaphase onset in control and p27/p25-depleted cells with and without p27 rescue plasmids.
Source data for this figure is available on the online supplementary information page.
